PROJECT SUMMARY Thymic development is highly responsible for shaping a healthy and balanced T cell immunity. Like other developmental processes it involves coordinated changes in the linear and three-dimensional chromatin organization that allow stage specific transcription events. A central regulator at nearly every stage of T cell differentiation is the DNA binding protein Tcf-1. Studies proposed here will elucidate how Tcf-1 coordinates the action of epigenetic and transcription regulators to instruct the differentiation of CD4+CD8+ DP thymocytes. Tcf- 1 modulates the chromatin landscapes and transcription profiles directly through binding to its conserved DNA sequence, or indirectly in association with other regulatory proteins. DP thymocytes express two forms of Tcf- 1, the full length Tcf-1p45 protein that binds ?-catenin, and a short Tcf-1p38 isoform that does not. Lef-1, another member of the Tcf/Lef family of regulators, also expressed in thymocytes has overlapping functions with Tcf-1. In addition Tcf-1 cooperates with the HLH domain DNA binding protein HEB at the DP thymocyte stages through the sharing of ~7000 DNA binding sites genome wide. The presence of both Tcf-1 and HEB at the shared sites is necessary to promote chromatin accessibility and regulate gene transcription. The direct Tcf-1- HEB binding to their conserved motifs in enhancer regions of T cell differentiation genes promotes their expression. By contrast Tcf-1-HEB recruitment to sites lacking conserved motifs, in promoter regions of cell- cycle genes reduces their expression and cell proliferation. Such opposing transcription outcomes likely involve Tcf-1-HEB recruitment to DNA in the context of distinct regulatory complexes. The composition of complexes containing Tcf-1 and HEB, the specific Tcf-1 isoform involved in each complex, and which functions are redundant between Tcf-1 and Lef-1, still remain to be elucidated. The intricate functional co-operation between these factors likely also involves the ability of Tcf-1 and Lef-1 to bend the DNA helix at their binding site which may modulate the 3D chromatin conformation, and define the proximity between co-operating factors and regulatory elements. These findings and the existing literature provide strong premise for the hypothesis that DP thymocyte development is enabled by the cooperation of Tcf-1 isoforms with protein complexes that shape the 3D chromatin structure to differentially regulate gene expression. Two specific aims are proposed. Aim 1 will determine the how Tcf-1p45 and, Tcf-1p33, co-operate with Lef-1 and HEB in the context of distinct regulatory complexes to establish the epigenetic and transcription profile of DP thymocytes and control their developmental progression. Aim 2: will elucidate the roles of Tcf-1p45, Tcf-1p33, Lef-1 with HEB shaping the chromatin conformation, and how this function promotes DP thymocyte development. The proposed studies are expected to highlight a completely new layer in the molecular regulation of DP thymocytes.